Oxidative stress induced by cumene hydroperoxide produces synaptic depression and transient hyperexcitability in rat primary motor cortex neurons.

Pyramidal neurons of the motor cortex are selectively degenerated in Amyotrophic Lateral Sclerosis (ALS). The mechanisms underlying neuronal death in ALS are not well established. In the absence of useful biomarkers, the early increased neuronal excitability seems to be the unique characteristic of ALS. Lipid peroxidation caused by oxidative stress has been postulated as one of the possible mechanisms involved in degeneration motor cortex pyramidal neurons. This paper examines the effect of lipid peroxidation on layer V pyramidal neurons induced by cumene hydroperoxide (CH) in brain slices from wild type rats. CH induces a synaptic depression of pyramidal neurons in a time dependent manner, already observable on GABAergic synaptic transmission after 5min application of the drug. Altogether, our whole-cell patch-clamp recording data suggest that the functional changes induced by CH upon pyramidal neurons are due to pre- and postsynaptic mechanisms. CH did not alter mEPSCs or mIPSCs, but decreased the frequency, amplitude, and decay rate of spontaneous EPSCs and IPSCs. These effects may be explained by a presynaptic mechanism causing a decrease in action potential-dependent neurotransmitter release. Additionally, CH induced a postsynaptic inward current that underlies a membrane depolarization. Depressing the input flow from the inhibitory premotor interneurons causes a transient hyperexcitability (higher resistance and lower rheobase) in pyramidal neurons of the motor cortex by presumably altering a tonic inhibitory current. These findings, which resemble relevant cortical pathophysiology of ALS, point to oxidative stress, presumably by lipid peroxidation, as an important contributor to the causes underlying this disease.

Time and dose dependent effects of oxidative stress induced by cumene hydroperoxide in neuronal excitability of rat motor cortex neurons.

It has been claimed that oxidative stress and the production of reactive oxygen radicals can contribute to neuron degeneration and might be one factor in the development of different neurological diseases. In our study, we have attempted to clarify how oxidative damage induces dose dependent changes in functional membrane properties of neurons by means of whole cell patch clamp techniques in brain slices from young adult rats. Our research demonstrates physiological changes in membrane properties of pyramidal motor cortex neurons exposed to 3 concentrations of cumene hydroperoxide (CH; 1, 10 and 100µM) during 30min. Results show that oxidative stress induced by CH evokes important changes, in a concentration and time dependent manner, in the neuronal excitability of motor cortex neurons of the rat: (i) Low concentration of the drug (1µM) already blocks inward rectifications (sag) and decreases action potential amplitude and gain, a drug concentration which has no effects on other neuronal populations, (ii) 10µM of CH depresses the excitability of pyramidal motor cortex neurons by decreasing input resistance, amplitude of the action potential, and gain and maximum frequency of the repetitive firing discharge, and (iii) 100µM completely blocks the capability to produce repetitive discharge of action potentials in all cells. Both larger drug concentrations and/or longer times of exposure to CH narrow the current working range. This happens because of the increase in the rheobase, and the reduction of the cancelation current. The effects caused by oxidative stress, including those produced by the level of lipid peroxidation, are practically irreversible and, this, therefore, indicates that neuroprotective agents should be administered at the first symptoms of alterations to membrane properties. In fact, the pre-treatment with melatonin, acting as an antioxidant, prevented the lipid peroxidation and the physiological changes induced by CH. Larger cells (as estimated by their cell capacitance) were also more susceptible to oxidative stress. Our results provide previously unavailable observations that large size and high sensitivity to oxidative stress (even at low concentrations) make pyramidal neurons of the motor cortex, in particular corticofugal neurons, more susceptible to cell death when compared with other neuronal populations. These results could also shed some light on explaining the causes behind diseases such as Amyotrophic Lateral Sclerosis.

Role for neonatal D-serine signaling: prevention of physiological and behavioral deficits in adult Pick1 knockout mice.

NMDA glutamate receptors have key roles in brain development, function and dysfunction. Regulatory roles of D-serine in NMDA receptor-mediated synaptic plasticity have been reported. Nonetheless, it is unclear whether and how neonatal deficits in NMDA-receptor-mediated neurotransmission affect adult brain functions and behavior. Likewise, the role of D-serine during development remains elusive. Here we report behavioral and electrophysiological deficits associated with the frontal cortex in Pick1 knockout mice, which show D-serine deficits in a neonatal- and forebrain-specific manner. The pathological manifestations observed in adult Pick1 mice are rescued by transient neonatal supplementation of D-serine, but not by a similar treatment in adulthood. These results indicate a role for D-serine in neurodevelopment and provide novel insights on how we interpret data of psychiatric genetics, indicating the involvement of genes associated with D-serine synthesis and degradation, as well as how we consider animal models with neonatal application of NMDA receptor antagonists.

Oxidative stress induced by cumene hydroperoxide evokes changes in neuronal excitability of rat motor cortex neurons.

Oxidative stress and the production of reactive oxygen radicals play a key role in neuronal cell damage. This paper describes an in vitro study that explores the neuronal responses to oxidative stress focusing on changes in neuronal excitability and functional membrane properties. This study was carried out in pyramidal cells of the motor cortex by applying whole-cell patch-clamp techniques on brain slices from young adult rats. Oxygen-derived free radical formation was induced by bath application of 10µM cumene hydroperoxide (CH) for 30min. CH produced marked changes in the electrophysiological properties of neurons (n=30). Resting membrane potential became progressively depolarized, as well as depolarization voltage, with no variations in voltage threshold. Membrane resistance showed a biphasic behavior, increasing after 5min of drug exposure and then it started to decrease, even under control values, after 15 and 30min. At the same time, changes in membrane resistance produced compensatory variations in the rheobase. The amplitude of the action potentials diminished and the duration increased progressively over time. Some of the neurons under study also lost their ability to discharge action potentials in a repetitive way. Most of the neurons, however, kept their repetitive discharge even though their maximum frequency and gain decreased. Furthermore, cancelation of the repetitive firing discharge took place at intensities that decreased with time of exposure to CH, which resulted in a narrower working range. We can conclude that oxidative stress compromises both neuronal excitability and the capability of generating action potentials, and so this type of neuronal functional failure could precede the neuronal death characteristics of many neurodegenerative diseases.

Postnatal development enhances the effects of cholinergic inputs on recruitment threshold and firing rate of rat oculomotor nucleus motoneurons.

Changes in the electrophysiological and morphological characteristics of motoneurons (Mns) of the oculomotor nucleus during postnatal development have been reported, however synaptic modifications that take place concurrently with postnatal development in these Mns are yet to be elucidated. We investigated whether cholinergic inputs exert different effects on the recruitment threshold and firing rate of Mns during postnatal development. Rat oculomotor nucleus Mns were intracellularly recorded in brain slice preparations and separated in neonatal (4-7 postnatal days) and adult (20-30 postnatal days) age groups. Stimulation of the medial longitudinal fasciculus evoked a monosynaptic excitatory potential in Mns that was attenuated with atropine (1.5 µM, a muscarinic antagonist). Mns were silent at their resting membrane potential, and bath application of carbachol (10 µM, a cholinergic agonist) induced depolarization of the membrane potential and a sustained firing rate that were more pronounced in adult Mns. Pharmacological and immunohistochemical assays showed that these responses were attributable to muscarinic receptors located in the membrane of Mns. In addition, compared to control Mns, carbachol-exposed Mns exhibited a higher firing rate in response to the injection of the same amount of current, and a decrease in the current threshold required to achieve sustained firing. These latter effects were more pronounced in adult than in neonatal Mns. In conclusion, our findings suggest that cholinergic synaptic inputs are already present in neonatal Mns, and that the electrophysiological effects of such inputs on recruitment threshold and firing rate are enhanced with the postnatal development in oculomotor nucleus Mns. We propose that cholinergic input maturation could provide a greater dynamic range in adult Mns to encode the output necessary for graded muscle contraction.

Temporal sequence of changes in electrophysiological properties of oculomotor motoneurons during postnatal development.

The temporal sequence of changes in electrophysiological properties during postnatal development in different neuronal populations has been the subject of previous studies. Those studies demonstrated major physiological modifications with age, and postnatal periods in which such changes are more pronounced. Until now, no similar systematic study has been performed in motoneurons of the oculomotor nucleus. This work has two main aims: first, to determine whether the physiological changes in oculomotor nucleus motoneurons follow a similar time course for different parameters; and second, to compare the temporal sequence with that in other neuronal populations. We recorded the electrophysiological properties of 134 identified oculomotor nucleus motoneurons from 1 to 40 days postnatal in brain slices of rats. The resting membrane potential did not significantly change with postnatal development, and it had a mean value of -61.8 mV. The input resistance and time constant diminished from 82.9-53.1 M omega and from 9.4-4.9 ms respectively with age. These decrements occurred drastically in a short time after birth (1-5 days postnatally). The motoneurons' rheobase gradually decayed from 0.29-0.11 nA along postnatal development. From birth until postnatal day 15 and postnatal day 20 respectively, the action potential shortened from 2.3-1.2 ms, and the medium afterhyperpolarization from 184.8-94.4 ms. The firing gain and the maximum discharge increased with age. The former rose continuously, while the increase in maximum discharge was most pronounced between postnatal day 16 and postnatal day 20. We conclude that the developmental sequence was not similar for all electrophysiological properties, and was unique for each neuronal population.

Afferent connectivity to different functional zones of the optic tectum in goldfish.

This work studies the afferent connectivity to different functionally identified tectal zones in goldfish. The sources of afferents contributed to different degrees to the functionally defined zones. The dorsocentral area of the telencephalon was connected mainly with the ipsilateral anteromedial tectal zone. At diencephalic levels, neurons were found in three different regions: preoptic, thalamic, and pretectal. Preoptic structures (suprachiasmatic and preoptic nuclei) projected mainly to the anteromedial tectal zone, whereas thalamic (ventral and dorsal) and pretectal (central, superficial, and posterior commissure) nuclei projected to all divisions of the tectum. In the mesencephalon, the mesencephalic reticular formation, torus longitudinalis, torus semicircularis, and nucleus isthmi were, in the anteroposterior axis, topographically connected with the tectum. In addition, neurons in the contralateral tectum projected to the injected zones in a symmetrical point-to-point correspondence. At rhombencephalic levels, the superior reticular formation was connected to all studied tectal zones, whereas medial and inferior reticular formations were connected with medial and posterior tectal zones. The present results support a different quantitative afferent connectivity to each tectal zone, possibly based on the sensorimotor transformations that the optic tectum carries out to generate orienting responses.

Connectivity of the goldfish optic tectum with the mesencephalic and rhombencephalic reticular formation.

The optic tectum of goldfish, as in other vertebrates, plays a major role in the generation of orienting movements, including eye saccades. To perform these movements, the optic tectum sends a motor command through the mesencephalic and rhombencephalic reticular formation, to the extraocular motoneurons. Furthermore, the tectal command is adjusted by a feedback signal arising from the reticular targets. Since the features of the motor command change with respect to the tectal site, the present work was devoted to determining, quantitatively, the particular reciprocal connectivity between the reticular regions and tectal sites having different motor properties. With this aim, the bidirectional tracer, biotin dextran amine, was injected into anteromedial tectal sites, where eye movements with small horizontal and large vertical components were evoked, or into posteromedial tectal sites, where eye movements with large horizontal and small vertical components were evoked. Labeled boutons and somas were then located and counted in the reticular formation. Both were more numerous in the mesencephalon than in the rhombencephalon, and ipsilaterally than contralaterally, with respect to the injection site. Furthermore, the somas showed a tendency to be located in the area containing the most dense labeling of synaptic endings. In addition, labeled boutons were often observed in close association with retrogradely stained neurons, suggesting the presence of a tectoreticular feedback circuit. Following the injection in the anteromedial tectum, most of the boutons and labeled neurons were found in the reticular formation rostral to the oculomotor nucleus. Conversely, following the injection in the posteromedial tectum, most of the boutons and neurons were also located in the caudal mesencephalic reticular formation. Finally, boutons and neurons were found in the rhombencephalic reticular formation surrounding the abducens nucleus. They were more numerous following the injection in the posteromedial tectum. These results demonstrate characteristic patterns of reciprocal connectivity between physiologically different tectal sites and the mesencephalic and rhombencephalic reticular formation. These patterns are discussed in the framework of the neural substratum that underlies the codification of orienting movements in goldfish.

Neural substrata underlying tectal eye movement codification in goldfish.

The optic tectum encodes orienting eye saccades in a spatially ordered map. To investigate whether the functional properties of each tectal site are related to a particular pattern of connectivity with downward structures in the brainstem, two sets of experiments were carried out. First, biotinylated dextran amine (BDA) was injected at different tectal sites along the anteroposterior axis. Electrical stimulation at these sites evoked saccades whose horizontal component amplitudes increased with the distance to the rostral pole. In the second experiment, BDA and fluoro-ruby (FR) were injected at different tectal sites along the mediolateral axis. Electrical stimulation here evoked saccades with different upward and downward directions, but similar horizontal component amplitudes. A major finding of the first experiment was that a topographic link of the tectum exists with the mesencephalic reticular formation, but that such a connection was absent or very attenuated for the rhombencephalic reticular formation. In the second set of experiments, the clusters of BDA and FR boutons left by the mediolateral tectal sites were separated in the rostral mesencephalon, at the level of the nucleus of the medial longitudinal fasciculus, but overlapped in the caudal mesencephalon and rhombencephalon. These data provide evidence that decodification of tectal motor commands is based, at least in part, on the connectivity of each tectal locus on downward structures with the brainstem.

Role of synaptic inputs in determining input resistance of developing brain stem motoneurons.

The contribution of synaptic input to input resistance was examined in 208 developing genioglossal motoneurons in 3 postnatal age groups (5-7 day, 13-16 day, and 18-24 day) using sharp electrode recording in a slice preparation of the rat brain stem. High magnesium (Mg(2+); 6 mM) media generated significant increases (21-38%) in both the input resistance (R(n)) and the first time constant (tau(0)) that were reversible. A large percent of the conductance blocked by high Mg(2+) was also sensitive to tetrodotoxin (TTX). Little increase in resistance was attained by adding blockers of specific amino acid (glutamate, glycine, and GABA) transmission over that obtained with the high Mg(2+). Comparing across age groups, there was a significantly larger percent change in R(n) with the addition of high Mg(2+) at postnatal days 13 to 15 (P13-15; 36%) than that found at P5-6 (21%). Spontaneous postsynaptic potentials were sensitive to the combined application of glycine receptor antagonist, strychnine, and the GABA(A) receptor antagonist, bicuculline. Application of either 10 microM strychnine or bicuculline separately produced a reversible increase in both R(n) and tau(0). Addition of 10 microM bicuculline to a strychnine perfusate failed to further increase either R(n) or tau(0). The strychnine/bicuculline-sensitive component of the total synaptic conductance increased with age so that this form of neurotransmission constituted the majority (>60%) of the observed percent decrease in R(n) and tau(0) in the oldest age group. The proportion of change in tau(0) relative to R(n) following strychnine or high magnesium perfusate varied widely from cell to cell and from age to age without pattern. Based on a model from the literature, this pattern indicates a nonselective distribution of the blocked synaptic conductances over the cell body and dendrites. Taken together, the fast inhibitory synapses (glycine, GABA(A)) play a greater role in determining cell excitability in developing brain stem motoneurons as postnatal development progresses. These findings suggest that synaptically mediated conductances effect the membrane behavior of developing motoneurons.

Role of potassium conductances in determining input resistance of developing brain stem motoneurons.

The role of potassium conductances in determining input resistance was studied in 166 genioglossal (GG) motoneurons using sharp electrode recording in brain stem slices of the rats aged 5-7 days, 13-15 days, and 19-24 days postnatal (P). A high magnesium (Mg(2+); 6 mM) perfusate was used to block calcium-mediated synaptic release while intracellular or extracellular cesium (Cs(+)) and/or extracellular tetraethylammonium (TEA) or barium (Ba(2+)) were used to block potassium conductances. In all cases, the addition of TEA to the high Mg(2+) perfusate generated a larger increase in both input resistance (R(n)) and the first membrane time constant (tau(0)) than did high Mg(2+) alone indicating a substantial nonsynaptic contribution to input resistance. With intracellular injection of Cs(+), GG motoneurons with lower resistance (<40 MOmega), on the average, showed a larger percent increase in R(n) than cells with higher resistance (>40 MOmega). There was also a significant increase in the effect of internal Cs(+) on R(n) and tau(0) with age. The largest percent increase (67%) in the tau(0) due to intracellular Cs(+) occurred at P13-15, a developmental stage characterized by a large reduction in specific membrane resistance. Addition of external Cs(+) blocked conductances (further increasing R(n) and tau(0)) beyond those blocked by the TEA perfusate. Substitution of external calcium with 2 mM barium chloride produced a significant increase in both R(n) and tau(0) at all ages studied. The addition of either intracellular Cs(+) or extracellular Ba(2+) created a depolarization shift of the membrane potential. The amount of injected current required to maintain the membrane potential was negatively correlated with the control R(n) of the cell at most ages. Thus low resistance cells had, on the average, more Cs(+)- and Ba(2+)-sensitive channels than their high resistance counterparts. There was also a disproportionately larger percent increase in tau(0) as compared with R(n) for both internal Cs(+) and external Ba(2+). Based on a model by Redman and colleagues, it might be suggested that the majority of these potassium conductances underlying membrane resistance are initially located in the distal dendrites but become more uniformly distributed over the motoneuron surface in the oldest animals.

Physiological changes accompanying anatomical remodeling of mammalian motoneurons during postnatal development.

The development of respiratory motoneurons provides unique data that may be generalized to other mammalian motoneuron populations. Like other motoneurons, respiratory motoneurons undergo developmental changes in the shape of the action potential and their repetitive firing. The unique observations concern the postnatal change in the recruitment pattern of cat phrenic motoneurons that is correlated with a halving of mean input resistance, a stasis of growth in the cell membrane and a reduction in the complexity of the dendritic tree. A similar pattern of change was observed for hypoglossal motoneurons studied in rat brainstem slices. Without an increase in total membrane surface area, the decreased resistance must result from a reduced specific membrane resistance. Two mechanisms are proposed to explain this decrease in resistance: proliferation and redistribution of either synaptic inputs and/or potassium channels. Although there was a significant contribution of synaptic input in determining input resistance throughout postnatal development, it was the density of cesium- or barium-sensitive potassium conductances that differentiated low resistance from high resistance motoneurons. Low resistance motoneurons had more cesium- and barium-sensitive channels than their high resistance counterparts. Based on the variations in the relative changes observed in input resistance versus membrane time constant with these two potassium channel blockers (cesium and barium), it is proposed that the distribution of these potassium channels change with age. Initially, their distribution is skewed toward the dendrites but as development progresses, the distribution becomes more uniform across the motoneuron membrane. During postnatal development, the rapid decrease in input resistance results from a proliferation of potassium channels in the membrane and of synaptic inputs converging onto developing respiratory motoneurons while the membrane is being spatially redistributed but not expanded.

Tectotectal connectivity in goldfish.

The vertebrate optic tectum is a functionally coupled bilateral structure which plays a major role in the generation of motor commands for orienting responses. However, the characteristics of the tectotectal connectivity are unknown in fish, and have been reported only to a limited extent in other vertebrates. The purpose of the present study was to determine the anatomical basis underlying the functional coupling between tecta in goldfish, and to identify both similarities and differences to those features reported in other vertebrate species. The present experiments used the bidirectional tracer biotinylated dextran amine to map the distribution of labeled cells and synaptic boutons in the contralateral tectum following injections into identified tectal sites. Fibers that interconnect both tecta coursed through the tectal commissure. The cells of origin of these fibers, the tectotectal cells, and their synaptic endings were located in the deep layers, mainly in the strata periventricular and griseum central, respectively. Corresponding sites throughout the two tecta were interconnected in a symmetrical point-to-point fashion. The tectal commissure was composed of at least two distinct bundles of axons, which differed in their dorsoventral location, fiber diameter, and projection targets. The dorsal axons were tectotectal axons, they were thinner in diameter and profusely branched, and gave off en passant and terminal boutons in the deep layers of the contralateral tectum. The ventral axons were thicker in diameter, and formed the contralateral tectofugal-descending tract. Such fibers had few axon collaterals and boutons in the contralateral tectum. Boutons adjacent to retrogradely labeled tectotectal cells were very scarce. The data are discussed in terms of the coupling between tecta generating the motor commands required for orienting movements.

Relationship between membrane properties and cell size of developing rat genioglossal motoneurons studied in vitro.

Electrical properties and morphology of 21 genioglossal motoneurons were measured in a slice preparation of the rat brainstem at four different postnatal ages. The motoneurons labeled with neurobiotin were reconstructed and quantified in three-dimensional space. There was no strong correlation found between the input resistance or membrane time constant and the total membrane surface area. We conclude that there is no electrical property of these developing motoneurons that can accurately predict their anatomical size.

Pontomedullary efferent projections of the ventral respiratory neuronal subsets of the rat.

The pontomedullary trajectories of projections efferent from the ventral respiratory cell group were anterogradely labelled after discrete injections of Fluoro Ruby into three morphophysiologically identified subdivisions (Bötzinger complex, rostral inspiratory, and caudal expiratory cell groups). The anterogradely labelled varicosities were located in a variety of areas involved in cardiorespiratory function: other subdivisions of the ventral respiratory cell group, the parabrachial (medial, central, and external lateral), Kölliker-Fuse, and lateral paragigantocellular nuclei, A5, and perifacial areas. Although the target areas were similar for the three studied subdivisions, some differences of the location and densities of labelled varicosities were found. Anterogradely labelled fibre bundles were found bilaterally after all of the tracer injections. Three caudally efferent bundles passed through the ventral respiratory cell group, dorsal medullary, and paramedian reticular nuclei. A labelled fibre bundle also took an ascending route through the ventral respiratory cell group: it surrounded the facial nucleus, and then followed two different pathways, one coursing towards forebrain areas and the other to the parabrachial and Kölliker-Fuse complex. Bundles of efferent axons decussated mainly at medullary levels and to a lesser extent in the pons. In the contralateral medulla and pons these labelled fibre bundles followed pathways similar to those observed ipsilaterally. The three ventral respiratory neuronal subsets sent axonal projections through similar tracts, but within them they were topographically organized. The present data are discussed with respect to the circuitry involved in the mechanisms of cardiorespiratory and other visceral functions.

Morphology of developing rat genioglossal motoneurons studied in vitro: relative changes in diameter and surface area of somata and dendrites.

This study describes the postnatal change in size of motoneurons in the hypoglossal nucleus that innervate the genioglossus muscle. Such anatomical information is essential for determining the cellular mechanisms responsible for the changes observed in the electrical properties of these motoneurons during postnatal development. The cells analyzed here are part of an earlier study (Núñez-Abades et al. [1994] J. Comp. Neurol. 339:401-420) where 40 genioglossal (GG) motoneurons from four age groups (1-2, 5-6, 13-15, and 19-30 postnatal days) were labeled by intracellular injection of neurobiotin in an in vitro slice preparation of the rat brainstem and their cellular morphology was reconstructed in three-dimensional space. The sequence of postnatal dendritic growth can be described in two phases. The first phase, between birth (1-2 days) and 13-15 days, was characterized by no change in either dendritic diameter (any branch order) or dendritic surface area of GG motoneurons. However, maturation of the dendritic tree produced more surface area at greater distances from the soma by redistributing existing membrane (retracting some terminal branches). During the second phase, between 13-15 days and 19-30 days, the dendritic surface area doubled as a result of an increase in the dendritic diameter across all branch orders and a generation of new terminal branches. In contrast to the growth exhibited by the dendrites, there was little discernible postnatal growth of somata. At all ages, dendrites of GG motoneurons show the largest amount of tapering in the first-and second-order dendrites. The calculated dendritic trunk parameter deviated from a value 1.0, indicating that the dendritic tree of developing GG motoneurons cannot be modeled accurately as an equivalent cylinder. However, the value of this parameter increased with age. Strong correlations were found between the diameter of the first-order dendrite and the dendritic surface area, dendritic volume, combined dendritic length, and, to a lesser extent, the number of terminal dendrites in GG motoneurons. Correlations were also found between somal and dendritic geometry but only when data were pooled across all age groups. These data support earlier studies on kitten phrenic motoneurons, which concluded that postnatal growth of motoneurons was not a continuous process. Based on the fact that there was no growth in the first 2 weeks, the changes in the membrane properties described during this phase of postnatal development (e.g., decrease in input resistance) cannot be attributed to increases in the total membrane surface area of these motoneurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Brain stem projections by axonal collaterals to the rostral and caudal ventral respiratory group in the rat.

The location of neurons projecting by axonal collaterals to the rostral and caudal ventral respiratory group (VRG) regions was determined after discrete injections of Fast blue and Diamidino yellow into the physiologically identified rostral inspiratory VRG and the caudal expiratory VRG areas, respectively. In contrast with single fluorochrome labeled neurons found throughout the rostro-caudal extent of the medulla and pons (in a variety of areas known to have cardiorespiratory function), double-labeled neurons were located in discrete ponto-medullary regions. The largest number of the double-labeled neurons was counted within the peripheral facial area, lateral paragigantocellular nucleus, and the VRG region, ipsi- and contralaterally to the injected side. Only a few double-labeled neurons were found within the ventrolateral and intermediate subnuclei of the solitary tract, medial parabrachial, and Kölliker-Fuse nuclei. The possible physiological implications of this neuronal network have also been emphasized.

Morphology of developing rat genioglossal motoneurons studied in vitro: changes in length, branching pattern, and spatial distribution of dendrites.

The aim of this study is to describe the postnatal change in dendritic morphology of those motoneurons in the hypoglossal nucleus that innervate the genioglossus muscle. Forty genioglossal (GG) motoneurons from four age groups (1-2, 5-6, 13-15, and 19-30 postnatal days) were labeled by intracellular injection of neurobiotin in an in vitro slice preparation of the rat brainstem and were reconstructed in three-dimensional space. The number of primary dendrites per GG motoneuron was approximately 6 and remained unchanged with age. The development of these motoneurons from birth to 13-15 days was characterized by a simplification of the dendritic tree involving a decrease in the number of terminal endings and dendritic branches. Motoneurons lost their 6th-8th order branches, in parallel with an elongation of their terminal dendritic branches maintaining the same combined dendritic length. The elongation of terminal branches was attributed to both longitudinal growth and the apparent lengthening caused by resorption of distal branches. The elimination of dendritic branches tended to increase the symmetry of the tree, as revealed by topological analysis. Later, between 13-15 days and 19-30 days, there was a reelaboration of the dendritic arborization returning to a configuration similar to that found in the newborn. The length of terminal branches was shorter at 19-30 days, while the length of preterminal branches did not change, suggesting that the proliferation of branches at 19-30 days takes place in the intermediate parts of terminal branches. The three-dimensional distribution of dendrites was analyzed by dividing space into six equal volumes (hexants). This analysis revealed that GG motoneurons have major components of their dendritic tree oriented in the lateral, medial, and dorsal hexants. Further two-dimensional polar analysis (consisting of eight sectors) revealed a reconfiguration of the tree from birth up to 5-6 days involving resorption of dendrites in the dorsal, dorsomedial, and medial sectors and growth in the lateral sector. Later in development (between 13-15 days and 19-30 days), there was growth in all sectors, but of a greater magnitude in the dorsomedial, medial, and dorsolateral sectors.

In vitro electrophysiology of developing genioglossal motoneurons in the rat.

1. Experiments were performed to determine the change in membrane properties of genioglossal (GG) motoneurons during development. Intracellular recordings were made in 127 GG motoneurons from rats postnatal ages 1-30 days. 2. The input resistance (R(in)) and the membrane time constant (t(aum)) decreased between 5-6 and 13-15 days from 84.8 +/- 25.4 (SD) to 47.0 +/- 18.9 M omega (P < 0.01) and from 10.0 +/- 4.2 to 7.3 +/- 3.3 ms (P < 0.05), respectively. During this period, the rheobase (Irh) increased (P < 0.01) from 0.13 +/- 0.07 to 0.27 +/- 0.14 nA, and the percentage of cells exhibiting inward rectification increased from 5 to 40%. Voltage threshold (Vthr) of the action potential remained unchanged postnatally. 3. There was also a postnatal change in the shape of the action potential. Specifically, between 1-2 and 5-6 days, there was a decrease (P < 0.05) in the spike half-width from 2.23 +/- 0.53 to 1.45 +/- 0.44 ms, resulting, in part, from a steepening (P < 0.05) of the slope of the falling phase of the action potential from 21.6 +/- 10.1 to 32.9 +/- 13.1 mV/ms. The slope of the rising phase also increased significantly (P < 0.01) between 1-2 and 13-15 days from 68.4 +/- 31.0 to 91.4 +/- 44.3 mV/ms. 4. The average duration of the medium afterhyperpolarization (mAHPdur) decreased (P < 0.05) between 1-2 (193 +/- 53 ms) and 5-6 days (159 +/- 43 ms). Whereas the mAHPdur was found to be independent of membrane potential, there was a linear relationship between the membrane potential and the amplitude of the medium AHP (mAHPamp). From this latter relationship, a reversal potential for the mAHPamp was extrapolated to be -87 mV. No evidence for the existence of a slow AHP was found in these developing motoneurons. 5. All cells analyzed (n = 74) displayed adaptation during the first three spikes. The subsequent firing pattern was classified into two groups, adapting and nonadapting. Cells at birth were all adapting, whereas all cells but two from animals 13 days and older were nonadapting. At the intermediate age (5-6 days), the minority (27%) was adapting and the majority (73%) was nonadapting. 6. The mean slope of primary range for the first interspike interval (1st ISI) was approximately 90 Hz/nA. This value was similar for both adapting and nonadapting cells and did not change postnatally.(ABSTRACT TRUNCATED AT 400 WORDS)

Brainstem connections of the rat ventral respiratory subgroups: afferent projections.

Propriobulbar neurons having axonal projections to the Ventral Respiratory Group (VRG) were retrogradely labeled after discrete injections of Fast blue into one of the three physiologically identified subdivisions (Bötzinger Complex, rostral inspiratory and caudal expiratory regions). Neurons that project to these regions were found throughout the rostrocaudal extent of the medulla and the pons in a variety of areas known to have cardio-respiratory function. Labeled somata were located within the nuclei of the solitary tract (commissural, intermediate and ventrolateral), other subdivisions of VRG, parabrachial nuclei (medial, dorsolateral and central lateral), Kölliker-Fuse nucleus, retrotrapezoid nucleus, lateral paragigantocellular nucleus and lateral tegmental field of the pons. Within the nuclei of the solitary tract and the Kölliker-Fuse nucleus, there was a topographical organization with respect to the three subdivisions of the VRG.